Expression of proteins and protein domains on the surface of self-replicating carriers such as bacteriophages, bacteria, yeasts, etc., is currently under intense investigation. The primary objective is to achieve coupling of the functional expression of a property of the protein exposed on the surface of the carrier (phenotype) with the fundamental genetic coding (genotype). Examples of successful phenotype/genotype coupling appear, among others, in the use of yeasts as carriers of surface-exposed proteins. For instance, a molecular library of variant yeast cells is generated, each one of which has different immunoglobulin fragments exposed on its cell surface. Those cells which exhibit increased affinity to a specific ligand molecule can be isolated from that library of antibody variations (Boder and Wittrup (1997), Nat. Biotechnol. 15:1553).
Bacteria have broad applications for cell surface exposure. Both Gram-positive and Gram-negative types are used. For instance, proteins can be exposed on Staphylococcus xylosus and Staphylococcus carnosus by Staphylococcus aureus protein A. Enzymes can be anchored to the surface of S. carnosus by fusion to Staphyloccus aureus fibronectin binding protein B (FnBPB) (Strauss and Götz (1996), Mol. Microbiol. 21:491–500). However, Gram-positive bacteria are less suitable for exposure of large peptide libraries because it is difficult to introduce the corresponding gene variants into the cells by transformation and to generate a sufficiently large number of independent clones which differ with respect to the coding nucleotide sequence of the surface-exposed protein variants.
Gram-negative bacteria, on the other hand, are very well suited to generation of molecular libraries and to the accompanying exposure of altered proteins because of their high transformation yield (>10° per microgram of plasmid DNA for E. coli), and so are preferred host organisms.
Various systems have been described for exposing recombinant proteins on the cell surface of Gram-negative bacteria (Georgiou et al. (1997), Nature Biotechnol. 15:29–34). In general, surface exposure is attained by fusing gene segments of a bacterial surface protein with the gene for the protein to be exposed. The proteins usually used as carriers are those which are secreted and/or localized in the external membrane of Gram-negative bacteria and therefore contain the signals needed for translocation through the cytoplasmic membrane, passage into the bacterial periplasm, and integration into the external membrane or anchoring on the surface of the external membrane. The carrier proteins that have been used most are those which are themselves integral components of the external membrane of E. coli. Those include, among others, PhoE (Agterbert et al. (1987), Gene 59:145–150) or OmpA (Francisco et al. (1992), Proc. Natl. Acad. Sci. USA 89:2713–2717); but there are disadvantages to their use. For instance, protein sequences can be inserted only into surface-exposed loops of these proteins. That results in conformationally fixed amino and carboxy terminations, and drastically limits the length of the peptide sequence to be inserted. Use of the peptidoglycan-associated lipoprotein (PAL) as a carrier protein does indeed result in transport to the external membrane, but it is impossible to expose active and correctly folded protein sequences on the surface of E. coli (Fuchs et al. (1991), Bio. Technology 9, 1369–1372). It has been possible to expose large proteins on the surface by (a) use of fusion of a fragment of the Escherichia coli Lpp and of the OmpA protein as the carrier protein portion, to the carboxy end of which the passenger protein sequence is attached (Francisco et al. (1992), Proc. Natl. Acad. Sci. 89:2713–2717); (b) use of the IgA protease (domain (IgAβ) and other bacterial autotransporters (Maurer et al. (1997) J. Bacteriol. 179: 794–804), and (c) by use of the ice nucleation protein of Pseudomonas syringae (InaZ) (Jung et al. (1998), Nature Biotechnol. 16:576–580 as the carrier protein portion.
It is clear from the examples above that proteins can be exposed on the bacterial cell surface by joining a passenger domain to a carrier protein by fusion of the corresponding coding DNA sequence with the coding sequence of a selected protein of the external membrane or membrane protein fragment. In this case the membrane protein or membrane protein fragment provides the force needed for the membrane localization and anchoring. Here the carrier protein of the external membrane should (a) have a secretion signal that assures passage through the cytoplasmic membrane; (b) exhibit a localization signal for embedment into the external membrane; (c) appear on the cell surface in the highest possible number of copies; and (d) not have a negative effect on the structural and functional integrity and, in particular, the vitality of the host cell.
Substantial problems have been found, though, with the processes described at the state of the art for production of heterologous passenger proteins using proteins of the external membrane, particularly with respect to requirements (c) and (d). A high expression ratio and a high net accumulation in the external membrane are always accompanied by high mortality of the bacterial cells which expose them. For instance, strong over-expression of fusion proteins with Lpp-OmpA as the membrane anchor is lethal (Daugherty et al. (1999), Protein Eng. 12:613–21). High cell mortality was likewise described for use of the autotranporter of the IgA protease (IgAβ) (Wentzel et al. (1999), J. Biol. Chem. 274: 21037–21043). Jung et al. Introduced the ice nucleation protein of Pseudomonas syringae, which is a glycosyl-phosphatidylinositol anchored protein of the external membrane, into E. coli as a carrier protein for cell surface exposure of passenger proteins (Jung et al. (1998), Nature Biotechnol. 16: 576–580). This carrier protein does allow stable exposure of passengers on the surface of the external membrane; but the fusion proteins aggregate in clusters on the bacterial surface. That characteristic is undesirable for the purpose of selecting peptides and polypeptides with high affinity to a specific binding partner.
Aside from the proteins integral to the external membrane, other surface structures present on the cell surface, such as flagellae, pili, fimbriae, etc., have been used as carriers for exposure of passenger domains. Various peptides of Hepatitis B virus were stably expressed and exposed on the bacterial surface by use of flagellin, a subunit of the flagellum, as the carrier (Newton et al. (1989), Science 244: 70–72). However, as for use of fimbrin as a structural carrier protein, exposure of passenger domains remains limited to small peptides (Hedegaard et al. (1989), Gene 85: 115–124).
Technical Problems, and Their Solution by the Present Invention
The present invention is, therefore, based on the technical problem of providing carrier proteins, which do not result in the disadvantages stated above, especially with use of Escherichia coli. 
An optimal presentation procedure must meet the following requirements:    1. The peptide/protein to be exposed should preferably be anchored on the surface of a bacterial cell in the highest possible number of copies.    2. The peptide/protein exposure should not impair viability.    3. The number of peptide/protein molecules exposed on the surface per cell should be controllable within wide limits.
No method of bacterial surface exposure which meets these requirements in all points has yet been described.